Gupta Mradu et al. Int. Res. J. Pharm.

2013, 4 (3)

INTERNATIONAL RESEARCH JOURNAL OF PHARMACY

ISSN 2230 8407

www.irjponline.com
Research Article

STUDIES OF ANTI INFLAMMATORY, ANTIPYRETIC AND ANALGESIC EFFECTS OF

AQUEOUS EXTRACT OF TRADITIONAL HERBAL DRUG ON RODENTS
Gupta Mradu 1*, Banerjee Dalia 2, Mukherjee Arup 3
1
Department of Medicinal Pharmacology (Dravyaguna), Institute of Post Graduate Ayurvedic Education and Research,
Kolkata, India
2
Department of Medicinal Pharmacology (Dravyaguna), Institute of Post Graduate Ayurvedic Education and Research,
Kolkata, India
3
Department of Chemical Technology, University College of Science and Technology, Calcutta University, Kolkata, India
Email: mradu_gupta@hotmail.com
Article Received on: 20/01/13 Revised on: 09/02/13 Approved for publication: 11/03/13
DOI: 10.7897/2230-8407.04321
IRJP is an official publication of Moksha Publishing House. Website: www.mokshaph.com
All rights reserved.
ABSTRACT
Aqueous extract of combination of stems of Tinospora cordifolia, fruits of Emblica officinalis and rhizomes of Cyperus rotundus has been used as traditional
herbal drug in Indian medicine system for treatment of fever, body ache, joint pain and inflammation. The collected botanicals were subject to physiochemical,
pharmacognostical & phytochemical screening before animal experiments. After acute toxicity studies, anti-inflammatory effect was assessed using carrageen
induced paw oedema test and antipyretic effect using yeast induced pyrexia method. Tail immersion, hot plate and writhings test were used for determining the
analgesic properties. Phytochemical analysis revealed the presence of polyphenolic flavonoids, tannin and saponins. Significant anti-inflammatory, antipyretic
and analgesic properties were noticed in dose dependant manner after aqueous extract administration especially at 600 mg/kg dose. These test drug activities
were sustained and comparable to the standard drugs while exhibiting no acute toxicity. Aqueous extract of test drug possesses significantly high antiinflammatory, antipyretic and analgesic properties without any acute toxicity possibly due to presence of flavonoids.
Keywords: Tinospora cordifolia, Emblica officinalis, Cyperus rotundus, anti-inflammatory, antipyretic, analgesic

INTRODUCTION
The Ayurvedic system of treatment approaches the whole
problem of disease in a holistic fashion. It advocates
correcting the imbalance through a combination of diet,
exercise, regimen and herbal medicines to accelerate the
process of healing and bringing the body-mind-sense
complex to a normal natural state. According to WHO,
traditional medicine has established and proved itself to
possess promotive, preventive, curative and rehabilitative
roles. The Traditional Herbal Medicinal Products Directive is
changing the face of the botanicals extracts and supplements
sector in the world for prevention and curing of diseases on
the basis of scientific evaluation of evidence based Ayurvedic
drugs. The test drug has been selected from the renowned
Ayurvedic text Charak Samhita which is having the
combination of three ingredients in equal amounts - stems of
Guduchi (Tinospora cordifolia Willd), fruits of Amlaki
(Emblica officinalis Gaertn) and rhizomes of Mustak
(Cyperus rotundus Linn) 1, 2. It has been traditionally used
for the treatment of fever, pain and inflammation in clinical
practice but its aqueous extract has not yet been subject to
scientific pharmacological evaluation.
Emblica officinalis Gaertn (family Euphorbiaceae), locally
known as Amlaki or Amla is a medium sized deciduous
tree found throughout India. Dried fruit of the plant is brown
to blackish brown in colour with characteristic odour and
sour and astringent taste. Its fruits are a rich source of
Vitamin C, besides it also contains tannin, ellagic acid and
gallic acid, phyllembins. They are also used in diabetes,
anaemia, peptic ulcer, inflammation, skin diseases and
cardiac problems. The fruit juice has been reported to possess
lipid lowering and anti-atherosclerotic effect 3, 4.
Tinospora cordifolia belonging to the family of
Menispermaceae locally known as Guduchi is a glabrous
climbing plant mostly found in India, typically growing in

deciduous and dry forests. The succulent stem is creamy

white to grey in color, with deep clefts spotted with lenticels
often giving out aerial roots. The principal constituents
found in its stem are Tinosporin, Tinocordiside,
Tinocordifolioside, Cordioside and alkaloids like Berberine
& Palmatine. This plant is used in Ayurvedic practice for
treatment of various ailments like leprosy, fever, asthma,
jaundice, diabetes, skin infections, diarrhea and dysentery4, 5,
6
.
Cyperus rotundus Linn (Cyperaceae), locally known as
Mustak or purple nutsedge or nutgrass, is a perennial weed
plant indigenous to India. Its rhizomes are bluntly conical and
vary in size and thickness, crowned with the remains of stem
and leaves forming a scaly covering, dark brown or black
externally, creamish-yellow internally and have pleasant
odour. The rhizomes are cooling, nervine tonic, and diuretic
and traditionally used to treat diarrhoea, leprosy, bronchitis
and blood disorders. The rhizome contains polyphenols like
Cyperone, Cyperenone and Cyperene and carbohydrates like
D-glucose and D-fructose. The rhizome is reported to possess
analgesic, anti-inflammatory and antipyretic properties 4, 7, 8.
The standard drugs used for analgesic, antipyretic and antiinflammatory action such as NSAIDS have been associated
with some side-effects and toxicity 9. Therefore, this
traditional drug which has been mentioned for the treatment
of fever, muscular pain, joint pain & inflammatory diseases
was taken up for its biological evaluation. Its aqueous extract
was subject to anti-inflammatory, antipyretic and analgesic
experimental studies carried out on rodents to compare its
efficacy with non-steroidal anti-inflammatory synthetic
drugs.
MATERIALS AND METHODS
All the experimental studies and chemical examinations were
performed in the laboratory and CPCSEA registered animal
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house facility located in the Dravyaguna department of the
Institute of Post Graduate Ayurvedic Education & Research,
Kolkata, India.
Collection & identification of plant samples
The samples of all the test botanicals in raw form were
collected from a reputed medicinal herb supplier of the
pharmacy of S. V. S. P. Hospital attached to IPGAER,
Kolkata. These samples were duly authenticated by the
Research Officer, Botanical Survey of India, Shibpur,
Howrah (Ref No- BSI/CNH/AD/Tech/2010 dated
21.07.2010) as per procedure laid down in the Ayurvedic
Pharmacopoeia of India 10.
Chemicals
Analytical grade chemicals for performing various types of
chemical and phytochemical analysis during this research
were purchased from a reputed company, namely M/s Merck
Pvt. Ltd.
Animals
The animal house & experimentation facility of IPGAER
which is registered with CPCSEA vide Reg. No
1180/ac/08/CPCSEA was used for all the experimental
studies. The requisite permissions were duly obtained from
the Institutional Animal Ethical Committee before
commencing the experiments. The anti-inflammatory and
antipyretic experiments were performed using adult Wistar
rats of either sex (1 month old and weighing 80-120gm)
while Swiss albino mice weighing 20-30 gm were used for
the acute toxicity study and the analgesic tests. All the
animals were procured from a reputed animal supplier of
Kolkata, Mr. Satyen Ghosh. The animals were housed in
polypropylene cages and maintained under environmentally
controlled room provided with a 12: 12 hr light and dark
cycle for each 24hr period at a temperature of approximately
25C. They were fed with standard pellet diet and water ad
libitum. Prior to experiments, the animals were fasted
overnight but allowed free access to water 11.
Extraction of Research drug
The ingredients of crude drugs were washed, sun dried and
crushed to particle size of 40 mesh. The powdered fruits of
Emblica officinalis, stems of Tinospora cordifolia, and
rhizomes of Cyperus rotundus were mixed in equal fraction
to prepare research drug. The research drug was then
subsequently extracted by Petroleum ether (60- 80),
Chloroform, Acetone, Methanol and Water using Soxhlet
Apparatus. The extracts obtained were filtered, concentrated
by rotary evaporator and finally stored in refrigerator for
further analysis. The aqueous extract of the research drug has
been used throughout the study 10, 12.
Pharmacognostical study of the crude drug powder
The macroscopic and microscopic examination of the test
drug powder was done according to standard procedures of
pharmacognosy. The final crude drug powder was mounted
in glycerine, observed under an optical microscope (40X) of
Dewinter, Italy and photographed.
The fluorescence analysis of the powder was done after
treating it with several solvents and then observing it under
Visible, UV 254 nm and UV 365 nm lamps.

Elemental analysis
Elemental analysis was performed to detect the presence of
nitrogen, sulfur and halogens using routine chemical analysis
techniques. A piece of metallic sodium was taken in a test
tube, melted by slow heating and about 0.5 g of research drug
powder was added which was strongly heated for about
2 min. Twenty ml of distilled water was taken in a mortar and
pastel, the red-hot test tube was broken and ground in mortar
distilled water. The aqueous solution was filtered through
Watman-40 filter paper and the filtrate was subjected to test
for these elements
Physiochemical Analysis
Physiochemical parameters such as extractive value, moisture
content, acid insoluble ash, water soluble ash and total ash
content of the powdered test drug were evaluated according
to standard steps described in the Ayurvedic Pharmacopoeia,
Government of India.
Phytochemical screening
Preliminary phytochemical screening for assessing the
presence of different active constituents like alkaloids,
flavonoids, tannins, carbohydrates, glycosides, saponins, fats
and oils, protein and amino acids was performed following
the standard procedures.
Estimation of total polyphenol content
Total polyphenol content was estimated using the Folin
Ciocalteu method calibrated on Gallic acid 20. Sample
extracts of 500 l were added to 500 l of water, 5 ml of
0.2 N FolinCiocalteu reagent and 4 ml of 75 g/l saturated
sodium carbonate solution and mixed in a cyclomixer. The
absorbance was measured in the spectrophotometer at
765 nm after incubation for 2 h at room temperature.
Quantification of total polyphenol content was done on the
basis of a standard curve generated with 100, 200, 300, 400
and 500 mg/l of Gallic acid 10, 12, 13.
Thin Layer Chromatography (TLC) analysis
The extract of the research drug (2.5 mg/ml) and standard
phenolic compound Gallic acid (0.6 mg/ml) were analysed
using a glass plate coated with a thin layer of 60 F254 silica
gel (194015 G, Batch No. HX024736, SISCO Research
laboratories Pvt. Ltd., Mumbai, India) using different solvent
mixtures. The development was stopped when the solvent
front had advanced about 7.5 cm. The different spots
developed were visualized on coloration (like Iodine vapour
exposure) and their Rf values were calculated. Rf values of
components are indicative of specific character of molecule
in the given environment of mobile and stationary phase. The
best results were obtained with the solvent system: Toluene:
Ethyl acetate: Formic acid: MeOH (3: 3: 0.8: 0.2 v/v/v/v).
Gallic acid was purchased from M/s Nice Chemicals Pvt. Ltd.
Experimental study
Acute toxicity study
Acute oral toxicity study was carried out according to OECD
guideline 423. Animals of both sexes were selected by
random sampling technique for the study and divided into 5
groups of 3 animals each. A single oral dose of the extract
starting at 200 mg/kg and progressively moving from 400,
600, 800 mg/kg up to 1000 mg/kg was administered. The
animal groups were observed for appearance of toxic
symptoms including behavioural changes, locomotion,
muscle spasm, loss of righting reflex, tremor, convulsions
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and mortality for 24 hrs and further supervised for a period of
14 days for occurrence of toxic symptoms and mortality 14.
Anti-inflammatory studies (Carrageenan - induced paw
oedema in rats)
The paw oedema was induced by injecting 0.1 ml of 1%
(w/v) Carrageenan suspension into the sub-planter region of
right hind paw of rats according to methods described by
Winter et al. (1962) [23]. The control group (A) was orally
administered saline (10 ml/kg) while the standard group (B)
was given Indomethacin (5 mg/kg) and Groups C & D were
given 400 mg/kg & 600 mg/kg of the test drug extract one
hour before Carrageenan injection 11, 15-18.
The measurement of paw oedema was carried out by
displacement technique using Plethysmometer to find out the
circumference of paw oedema immediately before and at 1hr,
2hr, 3hr and 4 hours following the Carrageenan injection.
The inhibitory activity was calculated according to the
formula% Inhibition= (Ct - Co) control (Ct - Co) treated x 100
(Ct - Co) control
where Ct is the paw circumference at time t, Co is the paw
circumference before Carrageenan injection and (Ct - Co) is
oedema or change in paw size after time t.
Evaluation of Antipyretic activity
The antipyretic efficacy of aqueous extract was assessed
using brewers yeast induced pyrexia method. Pyrexia was
induced by injecting 10.0 ml/kg of 20% w/v suspension of
Brewers yeast in normal saline subcutaneously 18 hours
before the commencement of experiment. Only animals
whose rectal temperature increased by at least 1.0C after 18
hours of induced subcutaneous yeast injection were included
in the study. The normal body temperature of each animal
was measured by inserting a flexible tail thermostat probe
coated with lubricant into rectum and recorded using digital
IMCORP Telethermometer. The experimental animals were
selected randomly and divided in four groups containing six
animals each. The control group (A) was orally administered
saline (10 ml/kg) while the standard group (B) was given 100
mg/kg Aspirin and Groups C & D were prescribed 400 mg/kg
& 600 mg/kg of the aqueous extract of test drug respectively.
The rectal temperature was recorded at time intervals of 1hr,
2hrs, 3hrs, 4hrs and 5 hours after drug administration 11, 16.
Evaluation of Analgesic activity
Assessment of central analgesic effect
Hot plate method
The central analgesic activity against thermal stimulus was
evaluated in mice following hot plate method as well as tail
immersion method. Morphine sulphate (2.5 mg/kg i.m.) was
used as the standard drug in the hot plate method. The control
group (A) was orally administered 10 ml/kg saline and the
standard group (B) was given intramuscular injection of 2.5
mg/kg Morphine. Similarly, Groups C & D were orally
administered 400 mg/kg & 600 mg/kg of the aqueous extract
of test drug respectively 1 hour before applying the thermal
stimulus, which was maintained at 55 0.2 oC. The latency
in hind paw licking was recorded in seconds as responses
after 10, 30 and 60 minutes of drug administration in the hot
plate method. Maximum reaction time of observation was
about 60 seconds throughout to avoid tissue damage 19, 20.

Tail immersion method

The tail immersion method was followed to find out the
central analgesic effect of aqueous extract in different
dosages 11, 21, 22. Morphine sulphate (2.5 mg/kg i.m.) was
used as the standard drug and injected into group B. The
control group, group C & group D were orally administered
10 ml/kg of saline, and 400 mg/kg & 600 mg/kg of the
aqueous extract of test drug respectively 1 hour before
applying the thermal stimulus by placing the tail 5 cm. in the
glass beaker having water maintained at 55 0.2 oC
temperature. The latency in tail withdrawal from the glass
beaker was recorded in seconds as response after 10, 30 and
60 minutes of drug administration in this method. Maximum
reaction time of observation was taken as about 60 seconds
throughout to avoid tissue damage 21, 22.
Assessment of peripheral analgesic effect (acetic acid
induced writhing analysis)
The peripheral analgesic activity of test drug was evaluated in
acetic acid induced writhing experiments using mice. The
abdominal constriction writhings resulting from intraperitoneal injection of acetic acid (10 ml/kg of 0.6% v/v
glacial acetic acid solution in water) were observed according
to standard procedure 22. 10 ml/kg Saline was orally
administered to group A (control group) whereas standard
Aspirin (100 mg/kg) was prescribed for group B and 400
mg/kg & 600 mg/kg of the aqueous extract of test drug was
orally given for Groups C & D respectively. Acetic acid
solution was administered after 30 minutes and number of
writhings counted in each animal for 15 minutes. Percentage
inhibition response was calculated as the reduction in the
number of abdominal constrictions between control group
and test drug treated groups as a percentage of the number of
writhes observed in case of the control group.
Statistical analysis
The differences in the parametric data were examined by
two-way analysis of variance (ANOVA) followed by
Dennetts t test, to compare a set of experimental data against
control mean. The level of significance was fixed between p
< 0.05 p < 0.01 23.
RESULTS
Pharmacognostical study
The observations under microscope showed the presence of
crystals and starch grains in large amount, cork cells,
raphides and parenchymatous cells in the powder.
Fluorescence analysis
The colours observed during the fluorescence analysis of the
drug sample in day light, UV 254 and UV 365 are outlined in
table 1.
Physiochemical analysis
The moisture content was observed to be 7.5 % w/w while
the pH was found to be 3.4. The total ash content was
estimated as 5.01 % w/w, the acid insoluble ash being 1.58 %
w/w and the water soluble ash content being 3.43 % w/w.
The extractive value (% w/w) in different solvent systems
was found to be 0.65% in petroleum ether, 0.69% in
Chloroform, 2.57% in Acetone, 4.1% in Methanol and 4.56%
in aqueous system.

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Table 1 Fluorescence analysis of drug sample
Reagent
1M Sodium Hydroxide
Acetic acid
1M Hydrochloric acid
Dil.Nitric acid
5% Iodine
5% Ferric Chloride
Methanol
50% Nitric acid
1M Sulphuric acid
Dil. Ammonia
Sodium Hydroxide in Methanol

Day light
light brown
Light brown
Light brown
Light greenish brown
Reddish black
Light green
Light brown
Creamish yellow
Light brown
Yellow
Yellow

Observation of colours
UV 254
Dark brown
Light brown
Greenish brown
Dark greenish brown
Dark brown
Dark green
Green
Green
Green
Dark green
Light green

UV 365
Blackish brown
Blackish brown
Blackish brown
Blackish red
Blackish red
Dark brown
Pesta
Black
Black
Black
Dark green

Table 2: Anti-inflammatory effect of Aqueous extract on carrageen induced paw oedema

Treatment group

0 hour

Control (10ml/kg)
Indomethacin (5mg/kg)
Aqueous extract (400mg/kg)
Aqueous extract (600mg/kg)

1hour

2 hour

3 hour

4hour

0.43 0.05
1.24 0.07
2.33 0.06
2.79 0.09
0.49 0.08
0.88 0.06
0.80 0.05
0.75 0.07
0.45 0.04
1.07 0.05
1.02 0.04
0.99 0.07
0.47 0.08
0.99 0.04
0.93 0.07
0.88 0.05
Results are presented as Mean SEM. p < 0.05 compared to control n=6

2.81 0.05
0.60 0.04
0.90 0.08
0.81 0.06

% inhibition
after 4hr
78.65
67.97
71.17

Table 3: Antipyretic effect of aqueous extract in comparison to Aspirin

Initial Rectal
Temp.(C)
Control

36.40 0.04

Standard

36.82 0.06

Aqueous Extract
(400mg/kg)
Aqueous extract
(600mg/kg)

36.51 0.02
36.63 0.03

Rectal Temperature in 0C after 18hrs of Yeast Injection (Mean SEM)

0 hr
1 hr
2 hr
3 hr
4 hr
37.22
37.62
37.53
37.4
37.31
0.07
0.03
0.05
0.03
0.02
37.99
37.36
37.18
37.12
36.97
0.04
0.02
0.05
0.04
0.05
37.12
36.88
36.82
36.79
37.7
0.05
0.06
0.03
0.02
0.07
37.87
36.89
36.75
36.72
36.7
0.02
0.04
0.03
0.04
0.05
Results are presented as Mean SEM. p < 0.05 compared to control n=6

5 hr
37.26
0.04
37.02
0.03
36.81
0.03
36.68
0.02

Reduction in
temperature after 4 hr
- 0.24%
2.69%
2.41%
3.09%

Table 4: Average reaction time using Hot plate method

Groups
Control
Standard Morphine
Aqueous Extract 400 mg/kg
Aqueous Extract 600 mg/kg

0 min
30 min
60 min
90 min
% increase in reaction time after 60 min
3.67 0.049
3.55 0.043
3.48 0.054
3.62 0.060
-5.18%
3.55 0.043
7.07 0.076
7.38 0.060
7.12 0.021
107.89%
3.35 0.022
5.45 0.060
5.66 0.048
5.94 0.033
68.96%
3.36 0.033
6.06 0.026
6.36 0.054
6.84 0.031
89.29%
Results are presented as Mean SEM. p < 0.05 compared to control n=6
Table 5: Average reaction time during Tail immersion method

Group
Control
Standard Aspirin
Aqueous Extract 400 mg/kg
Aqueous Extract 600 mg/kg

0 min
30 min
60 min
90 min
% increase in reaction time after 60 min
3.07 0.056
3.25 0.056
3.23 0.042
3.35 0.043
5.21%
3.27 0.049
6.68 0.130
6.75 0.056
6.27 0.080
106.42%
3.07 0.059
5.63 0.040
5.67 0.031
5.19 0.037
84.69%
3.05 0.043
5.82 0.021
5.92 0.033
5.40 0.043
94.09%
Results are presented as Mean+ SEM. p < 0.05 compared to control n=6
Table 6: Analgesic effect during Writhing method

Control
Standard (Aspirin)
Aqueous Extract 400 mg/kg
Aqueous Extract 600 mg/kg

Average number of Writhings /15 min

58.78 1.35
23.66 0.88
32.63 1.0 3
28.89 0.98

% Inhibition
0.0
59.78
44.53
50.89

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Figure 1: TLC plate obtained using the solvent system: Toluene: Ethyl
acetate: Formic acid: MeOH (3: 3: 0.8: 0.2)

Figure 2: Inhibition of acute inflammation in rat paws during carrageeninduced paw oedema test

Figure 3: Rectal temperature of rats during yeast induced pyrexia test

Figure 4: Reaction time using Hot plate method

Figure 5: Central analgesic effect during Tail immersion method

Figure 6: Peripheral analgesic effect in Writhing method

Elemental analysis
Only sulphur was found present in the research drug, while
nitrogen and halogens were found to be absent.

Estimation of total polyphenol content

The total phenol content was estimated to be 24.80 mg/gm of
dry mass as calculated from the standard curve of Gallic acid.

Thin Layer Chromatography (TLC)

After several runs in different solvent environments,
flavonoids in the hydrolyzate of the research drug moved
distinctly in flavonoid-specific solvent front Toluene: Ethyl
acetate: Formic acid: MeOH (3: 3: 0.8: 0.2) with Rf values of
0.344, 0.7444 and 0.967. Preliminary separation observed in
TLC plate (Figure 1) is suggestive of more than one
flavonidic components in the research drug.

Experimental Methods
Acute Toxicity study
The animals tested in acute toxicity study showed no
significant toxic symptoms up to the dose of 600 mg/kg such
as sedation, convulsion, diarrhoea, irritation, etc. At dosage
level of 1000 mg/kg, some animals showed mild symptoms
of irritation and minor behavioural changes but returned to
normal condition during a few hours. Even at this high dose,
no further toxic symptoms or mortality was observed for next
24 hrs and subsequently up to 14 days.
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Anti-inflammatory studies (Carrageenan-induced paw
oedema)
The circumference of paw oedema in rats induced by the
carrageen agent at 400 mg/kg and 600 mg/kg dose showed
significant decrease of 67.97% and 71.17% respectively (p
< 0.05) in inflammation up to 4 hours as compared to the
control group (table 2). The inhibition of acute inflammation
in the rat paws was found to be a little lower in 600 mg/kg of
test drug dosage (71.17% inhibition) as compared to the
standard drug Indomethacine (78.65% inhibition) after 4
hours of treatment (Figure 2).
Antipyretic effect of aqueous extract on yeast induced
pyrexia in rats
The yeast induced pyrexia experiment on rats showed an
average mean increase of about 1.11 C in rectal temperature
after 18 hrs of yeast injection. The test drug extract showed
significant antipyretic effect at both the doses (400 mg/kg and
600 mg/kg). The antipyretic effect of the extract at the dose
of 600 mg/kg is highly significant (p < 0.05) and comparable
to that of the standard drug, Aspirin. The test drug
administration resulted in a sharp decrease in the elevated
rectal temperature after 1hr of administration which was
further followed by a gradual trend of decrease in a dose
dependent manner. Unlike Aspirin and 400 mg/kg aqueous
extract which showed antipyretic activity up to 4 hrs, the
effect of the 600 mg/kg aqueous extract remained sustained
throughout the test period of 5 hrs. Thus, by the end of the 5th
hr of the experiment, the overall body temperature of rats in
the 600 mg/kg dose group almost came down to the initial
state while the animals in the other groups still showed an
elevated body temperature. The effect of the aqueous extract
of test drug on yeast induced pyrexia has been shown in table
3.
The overall reduction in the rectal temperature of animals
after 4 hours of oral drug administration was 2.69 % in case
of standard drug, 2.41 % in 400 mg/kg test drug and 3.09 %
in 600 mg/kg test drug while the control group showed an
increase of 0.24 % during this period. From the perspective
of percentage reduction of rectal temperature after 4 hours,
600 mg/kg dose of the test drug showed more pronounced
results as compared to the standard drug Aspirin. The test
drug also exhibited more sustained and persistent antipyretic
effect lasting up to 5 hours during this test (Figure 3).
Evaluation of Analgesic activity
Assessment of central analgesic effect
Hot plate method
The reaction time was increased significantly up to 60
minutes after giving the thermal stimulus in case of the 400
mg/kg drug group (increasing from 3.35 sec to 5.66 sec) &
600 mg/kg test drug group (increasing from 3.36 sec to 6.36
sec) when compared with the control group (where it actually
decreased from 3.67 sec to 3.48 sec) (table 4). The test drug
at the dose of 600 mg/kg showed similar type of pattern up to
90 minutes (reaction time increasing from 3.36 sec to 6.84
sec) but slightly lower central analgesic effect when
compared with the standard drug morphine sulphate (where
the reaction time increased from 3.55 sec to 7.12 sec) (Figure
4).
The results of the hot plate method showed that the
percentage increase in reaction time after oral administration
of drug and giving of thermal stimulus to the animals up to
60 minutes was 107.89 % in case of standard group, 68.96 %
in case of 400 mg/kg test drug group and 89.29 % in case of

600 mg/kg group. In comparison to this, the control group

actually showed a 5.18 % decrease in reaction time after 60
minutes. The test drug thus exhibited significant central
analgesic effect at both the doses of 400 mg/kg and 600
mg/kg as compared to the control group. However, the results
of test drug assessed in terms of the average reaction time at
the dose of 600 mg/kg (89.29% increase) showed a lower
central analgesic effect as compared with the standard drug
Morphine (107.89% increase).
Tail immersion method
Significant increase in reaction time was observed up to 60
minutes after giving thermal stimulus during the tail
immersion method in case of both the 400 mg/kg drug group,
where the increase was from 3.07 sec to 5.67 sec, and the 600
mg/kg test drug group where the reaction time went up from
3.05sec to 5.92 sec (table 5). In comparison, the reaction time
increased from 3.15 sec to only 3.35 sec in the control group.
During observations up to 90 minutes, a similar pattern was
observed the reaction time of 600 mg/kg test drug group
increased from 3.05 sec to 5.40 sec (Figure 5). However, its
overall central analgesic effect was a little lower when
compared with the standard drug Aspirin where the reaction
time increased from 3.27 sec to 6.27 sec during this period.
The increase in reaction time up to 60 minutes after oral
administration of drug and giving thermal stimulus in case of
tail immersion method was106.42 % in case of the standard
group, 84.69 % in case of the 400 mg/kg test drug group and
94.09 % in case of the 600 mg/kg group. The control group
showed only 5.21% increase in reaction time over the same
time period, indicating significant impact of the test drug and
standard drug during the experiment. The results indicated
that the test drug exhibited significant central analgesic effect
at both the doses of 400 mg/kg and 600 mg/kg as compared
to the control group. However, the overall impact of the test
drug assessed in terms of the average reaction time at the
dose of 600 mg/kg showed lower central analgesic effect as
compared to the standard drug Morphine.
Assessment of peripheral analgesic effect (acetic acid
induced writhing analysis)
The peripheral analgesic effect in the Writhing test was
evaluated on the basis of the average number of abdominal
constrictions indicated by the extension of hind paw of
animals. The observed inhibition in writhings as a result of
administration of the test drug was significantly higher (p <
0.05) at the dose of 400 mg/kg (44.53%) as well as at 600
mg/kg (50.89 %) when compared with the control group
(table 6). Comparing the performance of the test drug with
the standard drug, the observed peripheral analgesic effect
was slightly lower at 600 mg/kg test drug dose as indicated
by 50.89 % inhibition in writhings as compared to the
standard drug Aspirin which resulted in 59.78% inhibition
(Figure 6).
DISCUSSION
Inflammation is clinically defined as a patho-physiological
process characterized by redness, edema, fever, pain and loss
of function. Inflammation can result in locally increased
production of free radicals by inflammatory enzymes, as well
as the release of inflammatory mediators that promote cell
proliferation and angiogenesis and inhibit apoptosis.
Although the currently used steroidal anti-inflammatory
drugs (SAIDs) and non-steroidal anti-inflammatory drugs
(NSAIDs) treat acute inflammatory disorders, these
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conventional drugs have not been successful to cure chronic
inflammatory disorders such as rheumatoid arthritis (RA) and
atopic dermatitis (AD). Moreover, usage of many of such
drugs has been associated with adverse impact upon the
hepatic and renal functions 24, 25.
The preliminary chemical analysis of aqueous extract of this
botanical following standard method indicates high
concentration of phenolic compounds such as flavonoids,
tannins and carbohydrates. Flavonoids are a large family of
compounds synthesized by plants that have a common
chemical structure. The flavonoidic phenolic compounds
have been known to exhibit anti-inflammatory, antioxidant
and metal-chelating properties 9, 24.
The Hot plate and tail immersion methods indicated that the
central analgesic effect of the aqueous extract of test drug
was significant and dose dependent as revealed by the
increased reaction time after giving thermal stimulus to the
mice. The latency in reaction time continued up to 90 minutes
after the drug administration in both 400 mg/kg and 600
mg/kg extracts revealing sustained and pronounced central
analgesic effect when compared with the control group.
However, its overall central analgesic effect was a little lower
when compared with the standard drug Morphine over
different time periods, the higher test drug dosage resulting in
a more pronounced effect.
The assessment of peripheral analgesic effect of the test drug
exhibited significant percentage inhibition in the writhings
which were induced by acetic acid in the mice at both the 400
mg/kg and 600 mg/kg doses of the aqueous extract when
compared with the control group. The percentage inhibition
of writhings at the higher dose of 600 mg/kg indicated the
pronounced peripheral analgesic effect in the context of
visceral pain which was comparable to the standard pure drug
Morphine within 15 minutes of test. The significant analgesic
effect at the higher dose was attributed to the presence of
high concentration of flavonoidic compounds which inhibited
the synthesis, release or receptor responses in prostaglandin
mediated effects 9, 26, 27.
During antipyretic tests, the percentage reduction in rectal
temperatures after yeast injection up to 4 hours was quite
noticeable and comparable to the standard group in case of
600 mg/kg test drug. The observed antipyretic effect was
found to be sustained and lasted up to at least 5 hours after
oral administration of the drug sample in case of both the 400
mg/kg and 600 mg/kg aqueous samples which showed
similar pattern of antipyretic efficacy. Pyrexia is a result of
secondary impact of infection, tissue damage, inflammation,
graft rejection, malignancy or other diseased states.
Mediators like interleukin 1, , and TNF- increase the
synthesis of Prostaglandin E2 near pre-optic hypothalamus
area thereby triggering the hypothalamus to elevate the body
temperature. Flavonoids are known to target prostaglandins
which are involved in pyrexia 28, 29.
The evaluation of acute inflammation which was induced by
the carageenan in the paw of rats demonstrated the significant
percentage decrease in circumference and oedema resulting
from administration of the aqueous extract of test drug in
doses of 400 mg/kg and 600 mg/kg. The percentage
inhibition of acute inflammation in the paw of rats was found
to be lower in case of 600 mg/kg of test drug dosage when
compared to the standard drug Indomethacine after 4 hours of
treatment. The acute and sustained effect on the induced
inflammation up to four hours depends upon the decreased
production of pro-inflammatory cytokines and PGE2 in the
tissue of the effected part of the body. The inflammation

induced by phlogostic agent is related to the production of

histamine, bradykinin and cyclooxygenase products while
delayed phase is related to neutrophil infiltration, as well as
to the continuing of the production of arachinoic acid
metabolites. Prostaglandins and nitric oxide biosynthesis is
involved in inflammation, and isoforms of inducible nitric
oxide synthase (iNOS) and of cyclooxygenase (COX-2) are
responsible for the production of a great amount of these
mediators. It has been demonstrated that flavonoids are able
to inhibit both enzymes, as well as other mediators of the
inflammatory process such as reactive C protein or adhesion
molecules30.
The variety of flavonoids have been found to have
antimicrobial, antiviral, anti-ulcerogenic, cytotoxic, antineoplastic,
mutagenic,
antioxidant,
antihepatotoxic,
antihypertensive, hypolipidemic, antiplatelet and antipyretic
anti-inflammatory activities. Flavonoids also have
biochemical effects, which inhibit a number of enzymes such
as aldose reductase, xanthine oxidase, phosphodiesterase, Ca
(+2)-ATPase, lipoxygenase, cycloxygenase, etc 9, 31.
The detailed scientific evaluation of the pharmacological
properties namely the analgesic, antipyretic and antiinflammatory properties of the botanical medicine clearly
exhibit its therapeutic efficacy which was found to be
comparable to that of the standard drugs. The test drug was
found to be non toxic, significantly effective and having
sustained effect during this study. Many of these activities
could be attributed to the presence of flavonoids in the
aqueous abstract. However, further detailed studies are
needed to identify the responsible bio-chemical marker
compound in the test drug.
ACKNOWLEDGEMENTS
This research work was supported by grants from the Department of Science
and Technology, Government of West Bengal and we are grateful for that.
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Cite this article as:
Gupta Mradu, Banerjee Dalia, Mukherjee Arup. Studies of anti
inflammatory, antipyretic and analgesic effects of aqueous extract of
traditional herbal drug on rodents. Int. Res. J. Pharm. 2013; 4(3):113-120

Source of support: Nil, Conflict of interest: None Declared

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